This proposal is to develop a novel slide based proximity assay platform to quantify protein phosphorylation on tissues. The novelty of this assay platform is derived from the use of the quantum dot with two emission peaks for fluorescence resonance energy transfer (FRET): one emission as internal standard and the other as FRET donor. Change of ratio of the two emission intensity during the FRET can be used to quantify the ratio of two epitopes on a target protein. Phase I project demonstrated the feasibility of quantifying AKT and ERK phosphorylation on both cell lines and tissues. Phase II project will 1) complete validation of the quantification methods with patient derived animal model; 2) study c-Met protein phosphorylation; 3) validate the kits with PDX animal model, 4) establish quality control procedures and parameters and study the reproducibility and stability of the assays; and 5) establish GLP documentation of the assays. The final deliverable is four kits, AKT kit, ERK kit, c-Met kit and an universal kit.